We propose to address a hypothesis regarding the imaging and therapy of cancer with copper-64-labeled peptides, using somatostatin (SSTr2) ligands as well-characterized and model agents. The hypothesis is that the delivery of copper radionuclides from radiolabeled SSTr2 analogs to the nuclei of tumor cells will enhance their therapeutic efficacy. Although there might be several pathways to investigating mechanisms of cell death by copper radiopharmaceuticals, we will focus on whether differences in dissociation of Cu(II) from macrocyclic chelators in SSTR2 analogs (TETA- vs CB-TE2A-Y3-TATE) correlates to different nuclear localization in a SSTr2-positive tumor cell line (Aim 1). These data will be correlated to cell killing data in the same cell line (Aim 2). Aim 2 will also involve the estimation of absorbed doses to the cell nucleus from Cu-64-TETA-Y3-TATE and Cu-64-CB-TE2A-Y3-TATE. Specific Aim 3 will determine whether increased concentration of SSTr2 receptors correlates with increased internalization and subsequent nuclear localization in vitro, and increased uptake in SSTr2-transfected tumors with subsequent increased therapeutic efficacy in vivo. Specific Aim 4 will elucidate the mechanisms of trafficking of copper to the cell nucleus, in particular determining whether copper chaperone proteins are involved in delivery of copper from copper radiopharmaceuticals to the cell nucleus. Recently obtained data on 64Cu-labeled chelator-SSTr2 analogs has demonstrated the importance of 64Cu-chelate stability on normal organ uptake and clearance. We hope to correlate in vivo data with subcellular distribution, which will have implications for both imaging and therapy. We will delineate the importance of 64Cu-chelate stability as it relates to improving target: non-target tissue ratios, which are of great importance for imaging; however, the lack of chelate stability may enhance 64Cu localization to the cell nucleus which will enhance therapeutic efficacy. Defining this balance will help to shape the future of 64Cu radiopharmaceuticals, a rapidly growing area of research. The knowledge gained from this research will then be applicable to a wide variety of internalizing cell-surface receptor ligands for the development of new peptide or protein-based radiopharmaceuticals for cancer imaging and therapy.